The invention relates to the field of synthetic peptides derived from chemokines. In particular, the invention relates to antiviral agents useful for inhibiting the infectivity of human acquired immunodeficiency virus. More particularly, the present invention is directed to synthetic peptides capable of inhibiting HIV-1 infection.
Chemoattractant cytokines or xe2x80x9cchemokinesxe2x80x9d are a group of proteins characterized by a fairly high degree of amino acid sequence conservation. They are chemoattractants for leukocytes. The chemotactic effect of substances on leukocytes may be determined by devices such as the chemotaxis chamber invented by Boyden (Boyden, J. Ex. Med. 115:453-66, 1962).
Chemokines act on responsive leukocytes subsets through chemokine receptors. Engagement of chemokine receptors results ultimately in the movement of the cell. Chemokine receptors belong to the superfamily of G-protein coupled receptors (GPCR) that possess seven transmembrane helices (Murphy, P. M., Annu. Rev. Immunol. 12:593-633, (1994). The three-dimensional structure of chemokine receptors is not known since currently no crystal structure is available for any of GPCR proteins (Strader et al., Annu. Rev. Biochem. 63:101-32, (1994); Kobilka, B., Annu. Rev. Neurosci 15:87-114, (1992)).
Chemokines are the natural ligands for chemokine receptors and are 8-10 kDa molecules which act as chemoattractants by signaling through their receptors and activating the target cells (Premack et al., Nature Medicine 2:1174-8, (1996)). Chemokines may be divided into at least three structural branches: C, CC, and CXC, according to variations in a shared cysteine motif (Schall, Curr. Opin. Immunol. 6:865-873,1994). The CX, also known as a, and the CC, also known as xcex2, are the major classes of chemokines. Most CXC chemokines (those that contain an ELR sequence N-terminal to the CXC motif) are chemoattractants for neutrophils but not monocytes, whereas CC chemokines generally attract monocytes and lymphocytes, but not neutrophils. Basophils and eosinophils are affected predominantly by CC chemokines. The C chemokines appear to be lymphocyte specific.
Stromal-cell-derived factor-1 (SDF-1) is a member of the CXC chemokine family and is the ligand for CXC chemokine receptor 4 (CXCR4) (Tashiro et al., Science 261:600-603, (1993); Shirozu et al., Genomics28:495-500, (1995)). The CC chemokines MIP-1xcex2 (macrophage inflammatory protein 1xcex2), MIP-1a (macrophage inflammatory protein 1a) and RANTES (regulated on activation normal T cell expressed and secreted) bind CCR5.
Human immunodeficiency virus type 1 (HIV-1) enters cells through a fusion process in which the HIV-1 envelope glycoprotein gp 120 binds to CD4, the main receptor for HIV-1 on the cell surface. However, it has been known that CD4 alone is not sufficient for HIV-1 fusion and entry and that additional receptors may be needed (Maddon et al., Cell 47:333-348, (1986); Clapham et al., Virology 181:703-15, (1991)).
The chemokine receptors CXCR4 and CCR5 have been shown to be the long-sought coreceptors for non-syncytium-inducing and syncytium-inducing HIV-1 strains, respectively (Feng et al., Science 272:872-877, (1996); Deng et al., Nature 381:661-666, (1996); Dragic et al., Nature 381:667-673, (1996); Alkhatib et al., Science 272:1955-8, (1996)). While all HIV-1 strains appear to require either CXCR4, CCR5 or both (Zhang et al., Nature 383:768, (1996); Simmons et al., Journal of Virology 70:8355-60, (1996)), some strains can also use other chemokine receptors CCR3 and CCR2b as coreceptors for fusion and infection (Doranz et al., Cell 85:1149-58, (1996); Choe et al., Cell 85:1135-48, (1996)).
SDF-1 and CXCR4 play a role in HIV-1 viral entry. The fusion process may involve the initial binding of HIV-1 gp 120 to its high-affinity receptor CD4 which results in conformational changes in gp 120 and possibly also CD4 (Gershoni et al., Journal 7:1185-7, (1993); Clements et al., AIDS Research and Human Retroviruses 7:3-16. (1991); Sattentau et al., Journal of Virology 67:7383-93, (1993)). The gp 120-CD4 complex interacts with CXCR4 or other chemokine coreceptors to form a heterotrimeric complex of gp 120-CD4-coreceptor (Lapham et al., Science 274:602-5, (1996); Wu et al., Nature 6605:179-83, (1996); Trkola et al., Nature 384:184-7, (1996)). It has been shown that the HIV envelope can bind CXCR4 independently and that this interaction is enhanced by the presence of CD4 (Bandres et al., Journal of Virology 72:2500-2504, (1998).
On the other hand, it is also known that the CXCR4 ligand, SDF-1 inhibits HIV-1 infection (Bleul et al., Nature 382:829-833, (1996); Oberlin et al., Nature 382:833-835, (1996)).
Several inhibitors of HIV-1 have been found to target the coreceptor CXCR4 (Murakami et al., J. Exp. Med. 186:1389-133, (1997); Schols et al., J. Exp. Med. 186:1383-1388, (1997); Donzella et al., Nature Medicine 4:72-77, (1998); Doranz et al., J. Exp. Med. 186:1395-1400, (1997)). Synthetic peptides derived from SDF-1 have been shown to posses anti-HIV activity (Heveker etal., Current Biology 8:369-376 (1998)). While the solution structure of SDF-1 has been determined (Crump et al., EMBO J. 16:6996-7007 (1998)), there is no crystal structure available for CXCR4 to facilitate design of further inhibitors of HIV-1 binding to CXCR4.
There is a need for antiviral agents which can block HIV-1 entry via CXCR4. Preferably, but not necessarily, such peptides would not block the ability of CXCR4 to bind its natural ligand, SDF-1.
Ideally, selective inhibitors of HIV infection should comprise small molecule drugs. In contrast to other large protein-based therapeutics such as monoclonal antibodies, such small agents are advantageous since they are more likely to be non-immunogenic, orally administrable, and amenable for chemical synthesis and modification.
There is further need for improved chemokine peptides useful as therapeutic agents which provide enhanced activity and/or stability over existing chemokine peptides.
It is an object of the invention to provide antiviral synthetic peptides capable of inhibiting HIV-1 infection, by inhibiting HIV-1 mediated cytopathogenesis and cell fusion.
It is an object to provide a method of treating or inhibiting HIV-1 infection, by administration of the synthetic peptides.
It is an object of the invention to provide other synthetic chemokine peptides with enhanced properties.
The antiviral compounds of the present invention are in the form of peptides which possess anti-HIV activity. They inhibit HIV-1 entry into HIV-1 infection susceptible cells via the CXC chemokine receptor 4.
In all embodiments, the peptide may optionally comprise an amino-terminal and/or carboxy-terminal protecting group.
According to one embodiment, the invention is a peptide of the formula
X1-A-B-C-D-X2-E-F-G-X3-L-X4-J-K-M-N-P-Q-R-S-T-V-Ala-W-Y-X5xe2x80x83xe2x80x83(I)
wherein:
X1 is from zero to eight amino acids,
X2 is three amino acids,
X3 is from zero to eight amino acids,
L is a linker comprising a covalent bond or chemical moiety,
X4 is from zero to eight amino acids,
X5 is from zero to eight amino acids,
A is Ile, Leu, Phe or Val,
B is Ser or Thr
C is Phe, Tyr, or Trp,
D is Arg, His or Lys,
E is Arg, His or Lys,
F is Phe, Tyr, Trp or Leu
G is Phe, Tyr, Trp or Leu
J is Ile, Leu, Phe or Val,
K is Arg, Lys or His,
M is Phe, Tyr or Trp
N is Ile, Leu, Phe or Val,
P is Asn, Asp, Glu or Gin
Q is Asn, Asp, Glu or Gin
R is Tyr, Trp or Phe,
S is Ile, Leu, Phe or Val,
T is Asn, Asp, Glu or Gin
V is Arg, Lys or His,
W is Ile, Leu, Phe or Val,
Y is Asn, Asp, Glu or Gln, and said peptide optionally comprises an amino-terminal and/or carboxy-terminal protecting group.
In preferred embodiments, A is Leu, B is Ser, C is Tyr, D is Arg, E is Arg, F is Phe, G is Phe, J is Leu, K is Lys, M is Trp, N is Ile, P is Gln, Q is Glu, R is Tyr, S is Leu, T is Glu, V is Lys, W is Leu and/or Y is Asn.
In preferred embodiments, each of X1, X3, X4, and X5 is from zero to six amino acids, preferably zero to four amino acids.
In the foregoing embodiments of the invention, it should be understood that each amino acid of the segments X1, X2, X3, X4, and X5 may comprise, independent of any other amino acid in the segment, any amino acid.
According to another preferred embodiment,
X1 is
(i) zero amino acids, or
(ii) the segment A1-B1-C1-D1 or N-terminal truncation fragment thereof containing at least one amino acid, wherein:
A1 is Arg, Lys or His,
B1 is Pro or N-methylaianine,
C1 is Ile, Leu, Phe or Val, and
D1 is Ser or Thr;
X3 is
(i) zero amino acids, or
(ii) the segment A2-B2-2-D2-Ala-E2-Ala-F2, or C-terminal truncation fragment thereof containing at least one amino acid, wherein:
A2 is Asn, Asp, Glu or Gln,
B2 is Ser or Thr,
C2 is Arg, Lys or His,
D2 is Ile, Leu, Phe or Val,
E2 is Arg, Lys or His,
F2 is Asn, Asp, Glu or Gln;
X4 is
(i) zero amino acids, or
(ii) the segment A3-B3-C3-D3, or N-terminal truncation fragment thereof containing at least one amino acid, wherein:
A3 is Ile, Leu, Phe or Val,
B3 is Asn, Asp, Glu or Gln,
C3 is Pro or N-methylaianine,
D3 is Arg, Lys or His; and
X5 is zero amino acids.
According to another preferred embodiment, L is an amino acid sequence containing from 2 to 10 amino acids, more preferably from 2 to 6 amino acids, most preferably from 2 to 4 amino acids. In some embodiments of the invention, the amino acid sequence L comprises glycine or lysine amino acids.
In some embodiments, X2 is Cys-Pro-Cys.
According to other embodiments,
A-B-C-D is Leu-Ser-Tyr-Arg (SEQ ID NO:11),
E-F-G is Arg-Phe-Phe
J-K-M-N-P-Q-R-S-T-V is Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys (SEQ ID NO:12), and
W-Y is Leu-Asn.
According to other embodiments,
the segment A1-B1-C1-D1 is Lys-Pro-Val-Ser (SEQ ID NO:1) or N-terminal truncation fragment thereof containing at least one amino acid;
the segment A2-B2-C2-D2-Ala-E2-Ala-F2 is Glu-Ser-His-Val-Ala-Arg-Ala-Asn (SEQ ID NO:2), or C-terminal truncation fragment thereof containing at least one amino acid; and
the segment A3-B3-C3-D3 is Ile-Asp-Pro-Lys (SEQ ID NO:3), or C-terminal truncation fragment thereof containing at least one amino acid.
A pharmaceutical composition comprises a pharmaceutically acceptable carrier and a peptide according to the invention.
A method of inhibiting entry of HIV-1 into CXCR4-expressing cells comprises contact the cells with a peptide according to the invention.
A method of inhibiting infection by HIV-1 comprises administering to an individual an effective amount of a peptide according to the invention.
According to another embodiment of the invention, a synthetic chemokine peptide is provided. The peptide comprises an N-terminal segment comprising a sequence of from 10 to 25 amino acids having at least 50% sequence identity with a first reference segment of a naturally occurring chemokine, said first reference segment being found in the N-terminal region of said naturally occurring chemokine. The peptide further comprises a C-terminal segment comprising a sequence of from 10 to 25 amino acids having at least 50% sequence identity with a second reference segment of the naturally occurring chemokine, said second first reference segment being found in the C-terminal region of the naturally occurring chemokine. The N-terminal and C-terminal segments of the synthetic peptide are connected by a linker L as defined above. The linker links the C-terminus of the synthetic peptide N-terminal segment to the N-terminus of the synthetic peptide C-terminal segment. Preferably, the degree of sequence identity between each segment of the synthetic peptide and the corresponding first or second reference sequence of the naturally occurring chemokine is at least about 70%, more preferably at least about 80%, most preferable about 90%. In preferred embodiments, the amino acid sequence of the N-terminal segment differs, if at all, from the first reference segment of the naturally occurring chemokine only by conservative amino acid substitutions, and the amino acid sequence of the C-terminal segment differs, if at all, from the second reference segment of the naturally occurring chemokine only by conservative amino acid substitutions.
Other aspects and advantages of the present invention are described in the drawings and in the following detailed description of the preferred embodiments thereof.
xe2x80x9cCXCR4xe2x80x9d means CXC chemokine receptor 4.
xe2x80x9cHIV-1xe2x80x9d means human immunodeficiency virus type 1.
xe2x80x9cSDF-1xe2x80x9d means stromal cell-derived factor-1.
The nomenclature used to describe polypeptide compounds of the present invention follows the conventional practice wherein the amino group is presented to the left and the carboxy group to the right of each amino acid residue. In the formulae representing selected specific embodiments of the present invention, the amino-and carboxy-terminal groups, although not specifically shown, will be understood to be in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by a three-letter designation, corresponding to the trivial name of the amino acid, in accordance with the following schedule:
The following definitions, of terms used throughout the specification, are intended as an aid to understanding the scope and practice of the present invention.
A xe2x80x9cpeptidexe2x80x9d is a compound comprised of amino acid residues covalently linked by peptide bonds.
The expression xe2x80x9camino acidxe2x80x9d as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids. xe2x80x9cNatural amino acidxe2x80x9d means any of the twenty primary, naturally occurring amino acids which typically form peptides, polypeptides, and proteins. xe2x80x9cSynthetic amino acidxe2x80x9d means any other amino acid, regardless of whether it is prepared synthetically or derived from a natural source. As used herein, xe2x80x9csynthetic amino acidxe2x80x9d also encompasses chemically modified amino acids, including but not limited to salts, derivatives (such as amides), and substitutions. Amino acids contained within the peptides of the present invention, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the peptide""s circulating half life without adversely affecting their activity. Additionally, a disulfide linkage may be present or absent in the peptides of the invention, as long as anti-HIV activity is maintained.
Amino acids have the following general structure: 
Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group. Peptides comprising a large number of amino acids are sometimes called xe2x80x9cpolypeptidesxe2x80x9d. The amino acids of the peptides described herein and in the appended claims are understood to be either D or L amino acids with L amino acids being preferred.
As used herein, xe2x80x9cprotectedxe2x80x9d with respect to a terminal amino group refers to a terminal amino group of a peptide, which terminal amino group is coupled with any of various amino-terminal protecting groups traditionally employed in peptide synthesis. Such protecting groups include, for example, acyl protecting groups such as formyl, acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethane protecting groups such as benzyloxycarbonyl; and aliphatic urethane protecting groups, for example, tert-butoxycarbonyl or adamantyloxycarbonyl. See Gross and Mienhofer, eds., The Peptides, vol. 3, pp. 3-88 (Academic Press, New York, 1981) for suitable protecting groups.
As used herein, xe2x80x9cprotectedxe2x80x9d with respect to a terminal carboxyl group refers to a terminal carboxyl group of a peptide, which terminal carboxyl group is coupled with any of various carboxyl-terminal protecting groups. Such protecting groups include, for example, tert-butyl, benzyl or other acceptable groups linked to the terminal carboxyl group through an ester or ether bond.
By xe2x80x9csynthetic chemokine peptidexe2x80x9d is meant a peptide having an amino acid sequence, substantial portions of which are the same as or homologous to portions of the amino acid sequence of a naturally occurring chemokine.
By xe2x80x9cN-terminal segmentxe2x80x9d or xe2x80x9cN-terminal portionxe2x80x9d is meant a part of a peptide or mature polypeptide which terminates in the N-terminal amino acid of the peptide or polypeptide.
By xe2x80x9cC-terminal segmentxe2x80x9d or xe2x80x9cC-terminal portionxe2x80x9d is meant a part of a peptide or a mature polypeptide which terminates in the C-terminal amino acid or polypeptide.
By xe2x80x9cN-terminal truncation fragmentxe2x80x9d with respect to an amino acid sequence is meant a fragment obtained from a parent sequence by removing one or more amino acids from the N-terminus thereof.
By xe2x80x9cC-terminal truncation fragmentxe2x80x9d with respect to an amino acid sequence is meant a fragment obtained from a parent sequence by removing one or more amino acids from the C-terminus thereof.